PROJECT SUMMARY Type 1 diabetes is caused by the immune-mediated killing of pancreatic beta cells that produce insulin. The only long-term therapy available is insulin replacement. A number of patients have also benefited from islet transplantation and this transiently restores insulin sufficiency, but the availability of donor islets is extremely limiting. The recent development of methods to differentiate human stem cells into beta cells has made beta cell replacement a realistic prospect. Significantly, beta cells can be ?manufactured? from a patient?s own blood cells after induction of pluripotency and redifferentiation. This bypasses the problem of alloreactivity typical of organ transplantation. However, beta cells would still be destroyed after transplantation because of ongoing autoimmunity. To overcome this critical hurdle, we performed a genome-wide CRISPR-Cas9 screen to identify gene modifications that protect beta cells against immune-mediated killing. The experimental system we used, based on the NOD mouse model for type 1 diabetes, yielded only a dozen protective gene mutations out of more than 20,000 targeted genes. Remarkably, two of these candidate genes had previously been associated with human diabetes by GWAS. In this project, we will validate the lead candidate genes in experiments of increasing stringency and relevance to human diabetes. Our goal is to enable beta cell replacement in patients with type 1 diabetes by genome editing of stem cell-derived beta cells. To achieve this goal, we will pursue three specific aims. 1) We will validate candidate gene modifications for beta cell replacement in diabetic mice. Beta cells carrying candidate gene mutations, alone or in combination, will be tested for their capacity to resist immune-mediated killing in diabetic mice. We will first use an insulinoma beta cell line (NIT-1), then perform similar experiments with primary mouse islet cells. Once the most effective gene modifications are identified, we will perform beta cell replacement experiments using gene edited mouse islet cells to rescue diabetic mice with ongoing autoimmunity. 2) We will determine the mechanism by which protective gene modifications confer resistance to immune-mediated killing. Several gene mutations that protect against autoimmunity also protect against ER stress-mediated cell death. ER stress has been implicated in autoimmune diabetes owing to its effects on the post-translational modification of beta cell antigens. We will probe the unfolded protein response to understand how protective gene mutations modulate the beta cell response to ER stress as a possible pathway to immune evasion. 3) We will test the lead gene modifications in human stem cell-derived beta cells. We will introduce protective gene mutations into human stem cells and differentiate these into beta cells. Gene edited human cells will be tested for ER stress resistance and recognition by autoreactive T cells. We will also evaluate glucose stimulated insulin secretion of gene edited cells to ensure that protective mutations do not impede beta cell function. Collectively, these experiments will provide a stringent validation of protective gene modifications and lay a path to further preclinical testing.